need suggestions, protein isolation.

Achim Recktenwald, PhD ARecktenwald at StressGen.com
Mon Nov 29 14:52:19 EST 1999


Hi Robert,

1) Are you sure it is really a protein? 215 & 280nm absorption could also be
caused by non-proteinous compounds.

2) In case you are sure: after rp-HPLC, did you dry the sample in a SpeedVac
or a lyophilizer?
A possible cause for the loss of the protein might be drying the sample to
far down. Especially hydrophobic proteins/peptides or denatured ones - like
yours - concentrate them but do not dry them completely; always leave them
in a bit of liquid (10 - 30uL, maybe up to 100uL). Many hydrophobic proteins
cannot be re-dissolved again.


Cheers,

Achim


"hnasko" <hnaskor at email.uc.edu> wrote in message
news:81u7hv$o68$1 at news.ececs.uc.edu...
> November 29, 1999
>
> I hope someone may have some suggestions.  I have isolated a protein >5000
> MW <30,000 MW from cell conditioned media via RP-HPLC.  Sent the sample
off
> to a collaborator for sequence analysis and he cannot obtain data.  even
> post trypsin digest the MS gives only peaks of 500-900 Da.  Based on the
> chromatogram and the bioactivity of the sample I know it is there.  The
> magnitude of the peak at 215 and 280 nm suggests there is enough to get
> sequence 4x over.  We guess the problem is adsorption to the plastic
> (untreated tube).  This is the most likly, i would hate to think they dont
> know what they are doing.  Any suggestions as how we might get this
protein
> off the tube wall that is compatable with MS?  We are contemplating using
a
> detergent like SDS or TritonX followed by an HPLC step (scarry)?  We have
no
> structural info about this protein, only a bioactivity.  this tube
> represents 6.5 liters of material and lots of work, I hate to think it is
> lost.  We are going to restart purification with another 2 liters and are
> thinking of ways to treat our tubes to avoid adsorption problems, ie
> siliconization or teflon coated tubes.  I am not sure yet about the
> compatability of the silicon with the MS (any problems there?)  Any
> suggestions regarding these problems would be greatly appreciated.  Also
if
> you know a MS/protein core that is really good I am comtemplating trying a
> different group for another sample and would appreciate a reference I
could
> contact.
>
> thanx
>
> Robert Hnasko
> University of Cincinnati
> College of Medicine
> Dept. Cell biology, Neurobiology and Anatomy
>
> email:  hnaskor at email.uc.edu
>
>
>
>
>
>






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